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Abstract:

The present invention relates to novel, selective, radiolabelled PDE10
ligands which are useful for imaging and quantifying the PDE10A enzyme in
tissues, using positron-emission tomography (PET). The invention is also
directed to compositions comprising such compounds, to processes for
preparing such compounds and compositions, and to the use of such
compounds and compositions for imaging a tissue, cells or a host, in
vitro or in vivo.

Claims:

1. A compound of formula (I) ##STR00037## or a stereoisomeric form
thereof, wherein R1 is 2-fluoroethyl, 2,2,2-trifluoroethyl or
3-fluoropropyl; n is 1, 2 or 3; each R2 independently is
C1-3alkyl, cyclopropyl, C1-3alkyloxy, haloC1-3alkyloxy,
halo, trifluoromethyl, trifluoromethoxy, difluoromethoxy, or cyano,
wherein at least one F is [18F], or a solvate or a salt form thereof

2. The compound according to claim 1 wherein R1 is 2-fluoroethyl.

3. The compound according to claim 1 wherein R2 is 6-methyl,
3,5-dimethyl or 5-methoxy.

4. The compound according to claim 1 wherein the compound is
2-[[4-[1-(2-fluoroethyl)-4-(4-pyridinyl)-1H-pyrazol-3-yl]phenoxy]methyl]--
3,5-dimethyl-pyridine-succinate.

5. A sterile composition comprising a compound of Formula (I) as defined
in claim 1 dissolved in saline.

6. Use of a compound of formula (I) as defined in claim 1 for imaging a
tissue, cells or a host, in vitro or in vivo.

7. A method of imaging a tissue, cells or a host, comprising contacting
with or administering to a tissue, cells or a host a compound of Formula
(I) as defined in claim 1, and imaging the tissue, cells or host with a
positron-emission tomography imaging system.

8. A precursor compound of formula (VI) ##STR00038## or a
stereoisomeric form thereof, wherein m is 1 or 2; n is 1, 2 or 3; each
R2 independently is C1-3alkyl, cyclopropyl, C1-3alkyloxy,
C1-3alkyloxy, halo, trifluoromethyl, trifluoromethoxy,
difluoromethoxy, or cyano, or a solvate or a salt form thereof

Description:

FIELD OF THE INVENTION

[0001] The present invention relates to novel, selective, radiolabelled
PDE10 ligands which are useful for imaging and quantifying the PDE10A
enzyme in tissues, using positron-emission tomography (PET). The
invention is also directed to compositions comprising such compounds, to
processes for preparing such compounds and compositions, and to the use
of such compounds and compositions for imaging a tissue, cells or a host,
in vitro or in vivo.

BACKGROUND OF THE INVENTION

[0002] Phosphodiesterases (PDEs) are a family of enzymes encoded by 21
genes and subdivided into 11 distinct families according to structural
and functional properties. These enzymes metabolically inactivate widely
occurring intracellular second messengers, 3', 5'-cyclic adenosine
monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP).
These two messengers regulate a wide variety of biological processes,
including pro-inflammatory mediator production and action, ion channel
function, muscle contraction, learning, differentiation, apoptosis,
lipogenesis, glycogenolysis, and gluconeogenesis. They do this by
activation of protein kinase A (PKA) and protein kinase G (PKG), which in
turn phosphorylate a wide variety of substrates including transcription
factors and ion channels that regulate innumerable physiological
responses. In neurons, this includes the activation of cAMP and
cGMP-dependent kinases and subsequent phosphorylation of proteins
involved in acute regulation of synaptic transmission as well as in
neuronal differentiation and survival. Intracellular concentrations of
cAMP and cGMP are strictly regulated by the rate of biosynthesis by
cyclases and by the rate of degradation by PDEs. PDEs are hydrolases that
inactivate cAMP and cGMP by catalytic hydrolysis of the 3'-ester bond,
forming the inactive 5'-monophosphate (Scheme 1).

##STR00001##

[0003] On the basis of substrate specificity, the PDE families can be
divided into three groups: i) the cAMP-specific PDEs, which include PDE4,
-7 and -8; ii) the cGMP-selective enzymes PDE5 and -9; and iii) the
dual-substrate PDEs, PDE1, -2 and -3, as well as PDE10 and -11. The
discovery of phosphodiesterase 10A (PDE10A) was reported in 1999. Of all
the 11 known PDE families, PDE10A has most restricted distribution with
high expression only in the brain and testes. In the brain, PDE10A mRNA
and protein are highly expressed in the striatum. This unique
distribution of PDE10A in the brain, together with its increased
pharmacological characterization, points to the potential use of PDE10A
inhibitors for treating neurological and psychiatric disorders like
schizophrenia.

[0004] Positron Emission Tomography (PET) is a non-invasive imaging
technique that offers the highest spatial and temporal resolution of all
nuclear imaging techniques and has the added advantage that it can allow
for true quantification of tracer concentrations in tissues. It uses
positron emitting radionuclides such as, for example, 15O, 13N,
11C and 18F for detection.

[0006] Zhude Tu et al. disclose [11C]-papaverine as a PET tracer for
imaging PDE10A (Nuclear Medicine and Biology, 37, 509-516, 2010) and
conclude that it is not an ideal radioligand for clinical imaging of
PDE10A in the central nervous system. It is opined that analogues are
required having higher selectivity for PDE10A over PDE3 and improved
pharmacokinetic properties.

[0008] The present invention relates to a compound having the Formula (I)

##STR00002##

or a stereoisomeric form thereof, wherein R1 is 2-fluoroethyl,
2,2,2-trifluoroethyl or 3-fluoropropyl; n is 1, 2 or 3; each R2
independently is C1-3alkyl, cyclopropyl, C1-3alkyloxy,
C1-3alkyloxy, halo, trifluoromethyl, trifluoromethoxy,
difluoromethoxy, or cyano, wherein at least one F is [18F], or a
solvate or a salt form thereof.

[0009] The invention also relates to precursor compounds for the synthesis
of the compounds of Formula (I) as previously defined, said compounds
having the Formula (VI).

##STR00003##

or a stereoisomeric form thereof, wherein m is 1 or 2; n is 1, 2 or 3;
each R2 independently is C1-3alkyl, cyclopropyl,
C1-3alkyloxy, C1-3alkyloxy, halo, trifluoromethyl,
trifluoromethoxy, difluoromethoxy, or cyano, or a solvate or a salt form
thereof The invention also relates to reference compounds corresponding
to the [19F]-compounds of Formula (I).

[0010] Illustrative of the invention is a sterile composition comprising a
compound of Formula (I) described herein, dissolved in an appropriate
formulation.

[0011] Exemplifying the invention is a use of a compound of formula (I) as
described herein, for, or a method of, imaging a tissue, cells or a host,
in vitro or in vivo.

[0012] Further exemplifying the invention is a method of imaging a tissue,
cells or a host, comprising contacting with or administering to a tissue,
cells or a host a compound of Formula (I) as described herein, and
imaging the tissue, cells or host with a positron-emission tomography
imaging system.

[0013] Additionally, the invention refers to a process for the preparation
of a compound according to Formula (I) as described herein, comprising
the step of reacting a compound according to formula (V) as described
herein, with 11CH3I or 11CH3OTf in the presence of a
base in an inert solvent

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention is directed to compounds of Formula (I) as
defined hereinbefore and the pharmaceutically acceptable solvates and
salt forms thereof. The present invention is further directed to
precursor compounds of Formula (VI). In one embodiment, R1 is
2-fluoroethyl. In another embodiment R2 is 6-methyl, 3,5-dimethyl or
5-methoxy. In a preferred embodiment, the compound of formula (I) is
2-[[4-[1-(2-fluoroethyl)-4-(4-pyridinyl)-1H-pyrazol-3-yl]phenoxy]methyl]--
3,5-dimethyl-pyridine.succinate (B-3)

[0015] The compounds of Formula (I) and compositions comprising the
compounds of Formula (I) can be used for imaging a tissue, cells or a
host, in vitro or in vivo. In particular, the invention relates to a
method of imaging or quantifying PDE10A in a tissue, cells or a host in
vitro or in vivo.

[0016] The cells and tissues are preferably central nervous system cells
and tissues in which PDE10A is abundant. As already mentioned, PDE10A is
abundant in central nervous system tissue, more in particular, in central
nervous system tissue forming the brain; more in particular, forming the
striatum.

[0017] When the method is performed in vivo, the host is a mammal In such
particular cases, the compound of Formula (I) is administered
intravenously, for example, by injection with a syringe or by means of a
peripheral intravenous line, such as a short catheter.

[0018] When the host is a human, the compound of Formula (I) or a sterile
composition comprising a sterile saline solution of a compound of Formula
(I), may in particular be administered by intravenous administration in
the arm, into any identifiable vein, in particular in the back of the
hand, or in the median cubital vein at the elbow.

[0019] Thus, in a particular embodiment, the invention relates to a method
of imaging a tissue or cells in a mammal, comprising the intravenous
administration of a compound of Formula (I), as defined herein, or a
composition comprising a compound of Formula (I) to the mammal, and
imaging the tissue or cells with a positron-emission tomography imaging
system.

[0020] Thus, in a further particular embodiment, the invention relates to
a method of imaging a tissue or cells in a human, comprising the
intravenous administration of a compound of Formula (I), as defined
herein, or a sterile saline composition comprising a compound of Formula
(I) to the human, and imaging the tissue or cells with a
positron-emission tomography imaging system.

[0021] In a further embodiment, the invention relates to a method of
imaging or quantifying PDE10A in a mammal, comprising the intravenous
administration of a compound of Formula (I), or a composition comprising
a compound of Formula (I) to the mammal, and imaging with a
positron-emission tomography imaging system.

[0022] In another embodiment, the invention relates to the use of a
compound of Formula (I) for imaging a tissue, cells or a host, in vitro
or in vivo, or the invention relates to a compound of Formula (I), for
use in imaging a tissue, cells or a host in vitro or in vivo, using
positron-emission tomography.

Definitions

[0023] "C1-3alkyl", on its own or in combination with other terms
shall denote a straight or branched saturated alkyl group having 1, 2 or
3 carbon atoms, e.g. methyl, ethyl, 1-propyl and 2-propyl.

[0024] As used herein, the term "composition" is intended to encompass a
product comprising the specified ingredients in the specified amounts, as
well as any product which results, directly or indirectly, from
combinations of the specified ingredients in the specified amounts.

[0025] The term "stereoisomeric forms" as used hereinbefore or
hereinafter, defines all the possible stereoisomeric forms which the
addition salts of compounds according to formula (I) and their addition
salts may possess. Unless otherwise mentioned or indicated, the chemical
designation of compounds denotes the mixture of all possible
stereochemically isomeric forms.

[0026] Acceptable salts of the compounds of formula (I) are those wherein
the counterion is pharmaceutically acceptable. However, salts of acids
and bases which are non-pharmaceutically acceptable may also find use,
for example, in the preparation or purification of a pharmaceutically
acceptable compound. All salts, whether pharmaceutically acceptable or
not, are included within the ambit of the present invention. The
pharmaceutically acceptable salts are defined to comprise the
therapeutically active non-toxic acid addition salt forms that the
compounds according to Formula (I) are able to form. Said salts can be
obtained by treating the base form of the compounds according to Formula
(I) with appropriate acids, for example inorganic acids, for example
hydrohalic acid, in particular hydrochloric acid, hydrobromic acid,
sulphuric acid, nitric acid and phosphoric acid; organic acids, for
example acetic acid, hydroxyacetic acid, propanoic acid, lactic acid,
pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid,
fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic
acid, ethanesulfonic acid, benzensulfonic acid, p-toluenesulfonic acid,
cyclamic acid, salicylic acid, p-aminosalicylic acid and pamoic acid.

[0027] Conversely, said salt forms can be converted into the free base
form by treatment with an appropriate base.

[0028] The term "host" refers to a mammal, in particular to humans, rats,
mice, dogs and rats.

[0029] The term "cell" refers to a cell expressing or incorporating the
PDE10A enzyme.

[0030] The pyridine compounds of the present invention differ structurally
from the prior art compounds which invariably comprise a heteroaromatic
bicyclic moiety. Functionally they differ in that they have reduced
lipophilicity and, therefore, they exhibit less non-specific binding to
brain proteins and fat, making them more attractive as potential PET
radioligands. When compared to
2-{4-[1-(2-[18F]fluoroethyl)-4-pyridin-4-yl-1H-pyrazol-3-yl]-phenoxy-
methyl}-quinoline, the compounds of the present invention have faster
kinetics resulting in shorter acquisition times in clinical application
to obtain robust distribution volume values and reach higher
striatum-to-cerebellum ratios resulting in higher quality in vivo images
and more accurate quantification of the PDE10A binding potential.

Preparation

[0031] The compounds according to the invention can generally be prepared
by a succession of steps, each of which is known to the skilled person.
In particular, the compounds can be prepared according to the following
synthesis methods.

A. Preparation of the Final Compounds

[0032] Compounds of Formula (I) in their non-radiolabelled version can be
prepared by synthesis methods well known by the person skilled in the
art. Compounds of the invention may be prepared, for example, by three
different general methods:

Method A:

[0033] Following the reaction sequence shown in scheme 2.

##STR00004##

[0034] Thus, compound of Formula (II) may be reacted with a commercially
available alkylating agent of Formula (III), wherein Z is a suitable
leaving group such as halo, for example bromo or iodo, in the presence of
a suitable base such as cesium carbonate or potassium carbonate, in an
inert solvent such as, for example, dimethylformamide, stirring the
reaction mixture at a suitable temperature, typically at 100-150°
C., using conventional heating or under microwave irradiation, for the
required time to achieve completion of the reaction, typically 10-20
minutes in a microwave oven. The alkylation reaction usually affords a
mixture of the two possible regioisomers, derived from the alkylation on
both nitrogen atoms of the pyrazole ring, which can be separated by
chromatographic methods, either by column chromatography or HPLC.
Alternatively, Z may be a hydroxyl group, in which case reaction with
compound (II) can be performed using conventional Mitsunobu conditions,
which are well known by the person skilled in the art. Thus, compound
(II) can be reacted with compound (III) wherein Z is hydroxyl- in the
presence of diethyl-, di-tert-butyl- or diisopropyl azodicarboxylate and
triphenylphosphine, in an inert solvent such as for example
tetrahydrofuran, stirring the reaction mixture at a suitable temperature,
typically at 120° C. under microwave irradiation, for a suitable
period of time to allow completion of the reaction, typically 20 minutes.
The Mitsunobu reaction usually affords a mixture of the two possible
regioisomers, derived from the alkylation on both nitrogen atoms of the
pyrazole ring, which can be separated by chromatographic methods, either
by column chromatography or HPLC.

Method B:

[0035] Alternatively, compounds of formula (I) can also be prepared by a
reaction sequence as shown in scheme 3.

##STR00005##

[0036] Therefore, a compound of formula (IV) can be reacted with a
compound of formula (V) using conventional Mitsunobu conditions, which
are well known by the person skilled in the art. Thus, compound (IV) can
be reacted with compound (V) in the presence of diethyl-, di-tert-butyl-
or diisopropyl azodicarboxylate and triphenylphosphine, in an inert
solvent such as for example tetrahydrofuran, stirring the reaction
mixture at a suitable temperature, typically at 120° C. under
microwave irradiation, for a suitable period of time to allow completion
of the reaction, typically 15-20 minutes. Compounds of formula (V) are
either commercially available or are described in chemical literature and
can be prepared by simple standard synthetic procedures well known by the
skilled person.

Method C:

[0037] Alternatively, compounds of formula (I) wherein R1 is 2-fluoroethyl
or 3-fluoropropyl, may also be prepared by a reaction sequence as shown
in scheme 4.

##STR00006##

[0038] Therefore, in a compound of formula (VI) the hydroxyl group may be
transformed into a suitable leaving group LG, such as methanesulfonate or
tosylate, by methods well known by those skilled in the art, affording an
intermediate of formula (VII). Then the leaving group can be replaced by
fluorine using standard methods well known by the skilled person, such
as, for example, reacting with tetrabutylammonium fluoride in an inert
solvent such as for example tetrahydrofuran, stirring the reaction
mixture at a suitable temperature, typically at 70° C. under
microwave irradiation, for a suitable period of time to allow completion
of the reaction, typically 10 minutes. Alternatively, compounds of
formula (I) can also be prepared by direct reaction of an intermediate of
formula (VI) with a fluorinating agent such as, for example,
(N,N-diethylamino)sulphur trifluoride (DAST), by art known procedures.

[0039] The incorporation of radioactive fluorine atoms into the R1 side
chain of compounds of formula (I) wherein R1 is 2-fluoroethyl or
3-fluoropropyl, may be performed using techniques known in the art, for
example, by reaction of a suitable precursor of formula (VII) with a
nucleophilic radioactive fluorinating reagent, such as
K[18F]/Kryptofix® 222 or tetraalkyl ammonium salts incorporating
radioactive fluoride, in an inert solvent such as, for example,
dimethylformamide, stirring the reaction mixture at a suitable
temperature, using conventional heating or under microwave irradiation,
for the required time to achieve completion of the reaction.
Alternatively, the incorporation of radioactive fluorine can also be
performed by the alkylation reaction of an intermediate of formula (II)
with either 1-bromo-2-[18F]fluoroethane or
1-bromo-3-[18F]fluoropropane--in the presence of a base such as, for
example, cesium carbonate, in an inert solvent such as, for example,
dimethylformamide, stirring the reaction mixture at a suitable
temperature, using conventional heating or under microwave irradiation,
for the required time to achieve completion of the reaction, typically 15
minutes at 90° C. using conventional heating. In this case the
desired radiolabelled compound (I) can be separated from the other
radiolabelled regioisomer and from the unreacted precursor by HPLC.

[0040] The incorporation of either radioactive carbon atoms or radioactive
fluorine atoms into the R2 substituent groups of compounds of formula (I)
may also be performed using radiochemical techniques well known by those
skilled in the art. For example, a [11C]-methoxy group can be
incorporated by reaction of a suitable precursor of formula (I), wherein
one R2 is OH, with [11C]CH3I or [11C]CH3OTf in the
presence of a base, such as for example cesium carbonate, in an inert
solvent such as for example dimethylformamide, stirring the reaction
mixture at a suitable temperature using conventional heating-, for a
suitable period of time to allow completion of the reaction.

[0041] Incorporation of a radioactive fluorine atom in R2 can be achieved,
for example, by reaction of a suitable precursor of formula (I) wherein
one R2 is a nitro group, a chlorine or a bromine in either position 4 or
6 of the pyridinyl ring, with a nucleophilic radioactive fluorinating
reagent, such as K[18F]/Kryptofix® 222, in an inert solvent such
as, for example, dimethylformamide, dimethylsulfoxide or acetonitrile,
stirring the reaction mixture at a suitable temperature using
conventional heating or under microwave irradiation, for the required
time to achieve completion of the reaction.

[0042] The transformations of different functional groups present in the
final compounds, into other functional groups according to Formula (I),
can be performed by synthesis methods well known by the person skilled in
the art. Thus, for instance, a compound of formula (I) wherein R2 is a
bromine atom can react with (fluorosulfonyl)difluoroacetic acid methyl
ester, in the presence of cuprous iodide in an inert solvent such as, for
example, dimethylformamide, stirring the reaction mixture at a suitable
temperature, typically at 120° C., using conventional heating or
under microwave irradiation, for the required time to achieve completion
of the reaction, typically 2 h of conventional heating, to yield a
compound of formula (I) wherein R2 is trifluoromethyl. In another
example, a compound of formula (I) wherein R2 is a bromine atom can be
reacted with an alkyl- or cycloalkylboronic acid, in the presence of a
suitable base such as, for example, aqueous sodium carbonate and a
palladium complex catalyst such as, for example, palladium (0)
tetrakis(triphenylphosphine) in an inert solvent such as, for example,
dioxane, stirring the reaction mixture at a suitable temperature using
conventional heating or under microwave irradiation, for the required
time to achieve completion of the reaction, typically 15 minutes at
130° C. in a microwave oven, to yield a compound of formula (I)
wherein R2 is C1-3alkyl or cyclopropyl.

B. Preparation of the intermediate compounds

[0043] Intermediate compounds of formula (II) can be prepared by synthesis
methods well known by the person skilled in the art, such as, for
example, by the reaction sequence shown in scheme 5, which is based on
the method described in J. Med. Chem. 2009, 52 (16), 5188-5196.

##STR00007##

[0044] Therefore, a compound of Formula (VIII) may be reacted with excess
commercially available dimethoxymethyl-dimethylamine, stirring the
reaction mixture at reflux temperature for the required time to achieve
completion of the reaction, typically 1 hour. After evaporation to
dryness the resulting residue can be treated with hydrazine hydrate in
methanol, stirring the reaction mixture at reflux temperature for the
required time to achieve completion of the reaction, typically 1 hour.

[0045] Intermediate compounds of formula (VIII) can be prepared by a three
steps sequence reaction, following essentially the same synthesis method
as described in J. Med. Chem. 2009, 52 (16), 5188-5196, as it is shown in
scheme 6.

##STR00008##

[0046] Therefore, a compound of formula (IX) can be reacted first with
thionyl chloride, in order to form the corresponding acyl chloride, and
then in situ with O,N-dimethyl hydroxylamine, in the presence of a
suitable base such as, for example, triethylamine, in an inert solvent
such as, for example, tetrahydrofuran, stirring the reaction mixture at
room temperature for the required time to achieve completion of the
reaction, typically between 6 and 18 hours. The
N-methoxy-N-methyl-benzamide thus obtained can be subsequently reacted
with 4-picoline, in the presence of an organolithium reagent, typically
lithium diisopropylamide, in an inert and dry solvent such as, for
example, tetrahydrofuran, stirring the reaction mixture at -78° C.
for the required period of time to ensure completion of the reaction.

[0047] Intermediate compounds of formula (IX) can be prepared by synthesis
methods well known by the person skilled in the art, such as, for
example, by the reaction sequence shown in scheme 7.

##STR00009##

[0048] Therefore, a compound of formula (V) can be reacted first with
commercially available 4-hydroxybenzoic acid methyl ester, using
conventional Mitsunobu conditions, which are well known by the person
skilled in the art. Thus, compound (V) can be reacted with
4-hydroxybenzoic acid methyl ester in the presence of diethyl-,
di-tert-butyl- or diisopropyl azodicarboxylate and triphenylphosphine, in
an inert solvent such as for example tetrahydrofuran, stirring the
reaction mixture at a suitable temperature, typically at 120° C.
under microwave irradiation, for a suitable period of time to allow
completion of the reaction, typically 20 minutes. Subsequently, the
corresponding methyl ester derivative of compound (IX) thus obtained may
be hydrolized in basic conditions, using a diluted base such as, for
example, aqueous sodium hydroxide or potassium hydroxide in an inert
solvent such as, for example, methanol, tetrahydrofuran or a mixture
methanol/tetrahydrofuran, stirring the reaction mixture at a suitable
temperature, either at room temperature for a suitable period of time to
allow completion of the reaction, typically 18 hours, or at 150°
C. under microwave irradiation for 10 minutes. Compounds of formula (V)
are either commercially available or are described in chemical literature
and can be prepared by simple standard synthetic procedures well known by
the skilled person.

[0049] Intermediate compounds of formula (IV) can be prepared by synthesis
methods well known by the person skilled in the art, such as, for
example, by the reaction sequence shown in scheme 8.

##STR00010##

[0050] Therefore, a compound of formula (X) wherein Y is phenyl or
2-quinolinyl can be subjected to a hydrogenolysis reaction, in a suitable
inert solvent such as, for example, ethanol, in the presence of a
catalyst such as, for example, 5% or 10% palladium on activated carbon,
for a period of time that ensures the completion of the reaction,
typically at 50-80° C. and 1 atmosphere of hydrogen in an H-cube
apparatus.

[0051] Intermediate compounds of formula (X) can be prepared by synthesis
methods well known by the person skilled in the art, for example, in a
similar fashion as previously defined in method A for the synthesis of
compounds of formula (I), by the reaction sequence shown in scheme 9.

##STR00011##

[0052] Thus, a compound of formula (XI) wherein Y is phenyl or
2-quinolinyl may be reacted with a commercially available alkylating
agent of Formula (III), wherein Z is a suitable leaving group such as
halo, for example bromo or iodo, in the presence of a suitable base such
as cesium carbonate or potassium carbonate, in an inert solvent such as,
for example, dimethylformamide, stirring the reaction mixture at a
suitable temperature, typically at 100-150° C., using conventional
heating or under microwave irradiation, for the required time to achieve
completion of the reaction, typically 5-20 minutes in a microwave oven.
In the particular case when R1 is -CH2-CO-OAlk, the typical reaction
temperature is room temperature and the required time is 3 hours. The
alkylation reaction usually affords a mixture of the two possible
regioisomers, derived from the alkylation on both nitrogen atoms of the
pyrazole ring, which can be separated by chromatographic methods, either
by column chromatography or HPLC. Alternatively, Z may be a hydroxyl
group, in which case reaction with compound (XI) can be performed using
conventional Mitsunobu conditions, which are well known by the person
skilled in the art. Thus, compound (XI) can be reacted with compound
(III) wherein Z is hydroxyl in the presence of diethyl-, di-tert-butyl-
or diisopropyl azodicarboxylate and triphenylphosphine, in an inert
solvent such as for example tetrahydrofuran, stirring the reaction
mixture at a suitable temperature, typically at 120° C. under
microwave irradiation, for a suitable period of time to allow completion
of the reaction, typically 20 minutes. The Mitsunobu reaction usually
affords a mixture of the two possible regioisomers, derived from the
alkylation on both nitrogen atoms of the pyrazole ring, which can be
separated by chromatographic methods, either by column chromatography or
HPLC.

[0053] The synthesis of compound of formula (XI) wherein Y is phenyl is
described in patent application WO2006/072828. The synthesis of compound
of formula (XI) wherein Y is 2-quinolinyl is described in J. Med. Chem.
2009, 52 (16), 5188-5196.

[0054] Intermediate compounds of formula (VI) may be prepared by synthesis
methods well known by the person skilled in the art, such as, for
example, by the reaction sequence shown in scheme 10.

##STR00012##

[0055] Therefore, a compound of Formula (II) may be reacted with a
commercially available alkylating agent of Formula (XII), in which Z is a
suitable leaving group such as halo, bromo being the most preferred, in
the presence of a suitable base such as cesium carbonate or potassium
carbonate, in an inert solvent such as, for example, dimethylformamide,
stirring the reaction mixture at a suitable temperature, typically at
100° C., using conventional heating or under microwave
irradiation, for the required time to achieve completion of the reaction,
typically 10 minutes in a microwave oven. The alkylation reaction usually
affords a mixture of the two possible regioisomers, derived from the
alkylation on both nitrogen atoms of the pyrazole ring, which can be
separated by chromatographic methods, either by column chromatography or
HPLC.

[0056] Alternatively, compounds of formula (VI-a) wherein n is 1 can also
be prepared by the sequence of reactions shown in scheme 11.

##STR00013##

[0057] Thus, the ester group of a compound of formula (XIII) can be
reduced to alcohol by synthesis methods well known by the person skilled
in the art, such as, for example, reaction with sodium borohydride or
sodium cyanoborohydride, in a suitable inert solvent or mixture of
solvents, such as for example dichloromethane and methanol, stirring the
reaction mixture at a suitable temperature, typically at room
temperature, for the required time to achieve completion of the reaction,
typically 2 hours, to afford the compound of formula (VI-a) wherein n is
1.

[0058] Intermediate compounds of formula (XIII) can be prepared by
synthesis methods well known by the person skilled in the art, such as,
for example, by the reaction sequence shown in scheme 12.

##STR00014##

[0059] Thus, a compound of formula (II) may be reacted with commercially
available methyl- or ethyl bromoacetate, in the presence of a suitable
base such as cesium carbonate or potassium carbonate, in an inert solvent
such as, for example, dimethylformamide, stirring the reaction mixture at
a suitable temperature, typically at room temperature, for the required
time to achieve completion of the reaction, typically 3-6 hours, to yield
a compound of formula (XIII). The alkylation reaction usually affords a
mixture of the two possible regioisomers, derived from the alkylation on
both nitrogen atoms of the pyrazole ring, which can be separated by
chromatographic methods, either by column chromatography or HPLC.

[0060] Alternatively, an intermediate compound of formula (XIII) can also
be prepared by a reaction sequence as shown in scheme 13.

##STR00015##

[0061] Therefore, a compound of formula (IV-a) can be reacted with a
compound of formula (V) using conventional Mitsunobu conditions, which
are well known by the person skilled in the art. Thus, compound (IV-a)
can be reacted with compound (V) in the presence of diethyl-,
di-tert-butyl- or diisopropyl azodicarboxylate and triphenylphosphine, in
an inert solvent such as for example tetrahydrofuran, stirring the
reaction mixture at a suitable temperature, typically at 120° C.
under microwave irradiation, for a suitable period of time to allow
completion of the reaction, typically 15-20 minutes. The compound of
formula (IV-a) can be synthesized following the hydrogenolysis reaction
sequence that is shown above in scheme 8, starting from a compound of
formula (X-a) wherein R1 is --CH2-CO--OAlk, which can be synthesized
according to the reaction sequence shown above in scheme 9.

[0062] The activity of compounds of Formula (I) was determined by
measuring rat PDE10A2-cAMP inhibition and the pIC50 ranged from 6.60 to
8.79. The selectivity over other PDEs was also measured and it was in all
cases>50 fold and in most of the compounds>100 fold.

Applications

[0063] The compounds according to the present invention find various
applications for imaging tissues, cells or a host, both in vitro and in
vivo. Thus, for instance, they can be used to map the differential
distribution of PDE10 in subjects of different age and sex. Further, they
allow one to explore for differential distribution of PDE10 in subjects
afflicted by different diseases or disorders. Thus, abnormal distribution
may be helpful in diagnosis, case finding, stratification of subject
populations, and in monitoring disease progression in individual
subjects. The radioligands may further find utility in determining PDE10A
site occupancy by other ligands. As the radioligands are administered in
trace amounts, no therapeutic effect will result therefrom.

[0068] Several methods for preparing the compounds of this invention are
illustrated in the following examples, which are intended to illustrate
but not to limit the scope of the present invention. Unless otherwise
noted, all starting materials were obtained from commercial suppliers and
used without further purification.

[0071] A mixture of 6-methyl-2-pyridinemethanol (7.0 g, 56.84 mmol),
methyl 4-hydroxybenzoate (8.65 g, 56.84 mmol), diisopropyl
azodicarboxylate (14.65 mL, 73.9 mmol) and triphenylphosphine (19.38 g,
73.9 mmol) in THF (42 mL) was heated in a microwave oven at 120°
C. for 20 min (the reaction was divided in 7 batches). After this time
the mixture was quenched with water, extracted with DCM, the solvent was
evaporated to dryness in vacuo and the crude residue was purified by
column chromatography (silicagel; heptane/EtOAc from 80/20 to 50/50). The
desired fractions were collected and evaporated in vacuo to yield the
desired intermediate I-1 as an orange oil around 50% pure, the main
impurity being triphenylphosphine oxide. The mixture was used in the next
reaction step without further purification (25 g, 85.5%).
C15H15NO3.

Intermediate 2

[0072] 4-(6-Methyl-pyridin-2-ylmethoxy)-benzoic acid (I-2)

##STR00017##

[0073] To a solution of intermediate I-1 in 150 mL of a mixture of
methanol/THF (2:1) was added 2 M aqueous sodium hydroxide (49 mL, 97.2
mmol). The reaction mixture was stirred at room temperature overnight,
then at 60° C. for 2 h. After evaporation of the organic solvent
the aqueous phase was washed with EtOAc and then acidified with diluted
HCl to pH is 5-6. Intermediate compound 1-2 that precipitated was
filtered off, dried and used for the next reaction step without further
purification (9.0 g, 76.2%). C14H13NO3.

[0075] A mixture of intermediate 1-2 (9.0 g, 37.0 mmol) and thionyl
chloride (50 mL) was stirred at room temperature for 3 h. The mixture was
then concentrated to dryness and the crude acid chloride was dissolved in
THF (100 mL). Then triethylamine (20.5 mL, 148 mmol) and
O,N-dimethyl-hydroxylamine hydrochloride (10.8 g, 111 mmol) were slowly
added. The reaction mixture was stirred at room temperature overnight.
After quenching with water, the mixture was extracted with EtOAc, dried
over sodium sulphate, filtered and evaporated in vacuo. The residue was
purified by column chromatography (silicagel; heptane/EtOAc from 40/60 to
0/100) to give intermediate compound 1-3 as an orange oil that was used
for the next reaction without further purification (6.2 g, 46.8%).
C16H18N2O3.

[0077] To a solution of lithium diisopropylamide (2 M solution in THF,
43.3 mL, 86 6 mmol) in THF (35 mL) was added dropwise 4-methylpyridine
(8.43 mL, 86 6 mmol) at 0° C. under nitrogen. After 30 min it was
cooled down to -78° C. and 35 mL of this solution was added
dropwise to another solution of intermediate 1-3 (6.2 g, 17.32 mmol) in
THF (65 mL) also cooled to -78° C. The mixture was stirred at this
temperature for 2 h and then 20 mL more of the initially prepared
solution were added dropwise. The resulting solution was stirred at
-78° C. for an additional hour. After this time the reaction
mixture was quenched with water and extracted with DCM. The organic layer
was dried over sodium sulphate, filtered and evaporated in vacuo. The
crude residue obtained was washed and triturated with diethyl ether
affording intermediate compound 1-4 (4.7 g, 85.2%) as a pale yellow
solid. C20H18N2O2.

[0079] A solution of intermediate I-4 (4.7 g, 14.76 mmol) in
dimethoxymethyl-dimethylamine (15 mL) was stirred at reflux for 1 h.
After evaporation of the solvent the crude residue was dissolved in
methanol (50 mL) and hydrazine hydrate (1.08 mL, 22.14 mmol) was added.
The reaction mixture was heated at reflux for 1 h, after which time the
solvent was evaporated affording a solid residue that was washed and
triturated with a mixture of diethyl ether/EtOAc, to yield intermediate
compound 1-5 (3.4g, 67.3%) as a pale yellow solid.
C21H18N4O. LCMS: Rt 2.48, m/z 343 [M+H].sup.+ (method 7).

[0081] A mixture of 4-[3-(4-benzyloxy-phenyl)-1H-pyrazol-4-yl]-pyridine
(6.7 g, 20.47 mmol) that was synthesized following the method described
in patent application WO2006/072828, 1-bromo-2-fluoroethane (3.12 g,
24.56 mmol) and cesium carbonate (20 g, 61 4 mmol) in DMF (42 mL) was
heated in a microwave oven at 150° C. for 5 min (the reaction was
divided in 6 batches). After cooling to room temperature, the solid was
discarded by filtration and the solution was quenched with water and
further extracted with EtOAc. The organic phase was dried over sodium
sulphate, filtered and evaporated in vacuo. The crude residue was
purified by column chromatography (silicagel;
acetonitrile/diisopropylether from 30/70 to 80/20, and then again with
EtOAc/heptane 70/30). The desired fractions were collected and evaporated
to dryness yielding the desired intermediate compound 1-6 (4 g, 52.3%) as
an oil. C23H20FN3O. LCMS: Rt 4.22, m/z 374 [M +H].sup.+
(method 1).

[0092] Intermediate compound 1-9 was dissolved in ethanol (50 mL) and
subjected to hydrogenation in a H-Cube® system using Pd/C 5% as
catalyst (full hydrogen mode, 1.5 mL/min) at 80° C. and
atmospheric pressure. The solvent was evaporated to dryness in vacuo
affording intermediate compound I-10 (0.55 g, 75.4%) as a white solid,
which was used for the next reaction without further purification.
C16H12F3N3O. LCMS: Rt 1.82, m/z 320 [M+H].sup.+
(method 7).

[0094] To a stirred solution of intermediate 1-5 (0.30 g, 0.876 mmol) in
DMF (6 mL) were added methyl bromoacetate (0.10 mL, 1.051 mmol) and
cesium carbonate (0.86 g, 2.63 mmol). The mixture was stirred at room
temperature for 6 h. Then it was quenched with water and extracted with
EtOAc. The organic solvents were separated, dried over sodium sulphate
and evaporated to dryness in vacuo. The crude residue was purified by
column chromatography (silicagel; EtOAc/methanol 100/0 to 90/10). The
desired fractions were collected and evaporated in vacuo to yield a
mixture of the two regioisomers I-11 and I-11' that was used as such for
the next reaction without further purification (0.21 g, 49.2%).
C24H22N4O3.

[0099] To a stirred solution of
2-[4-(4-Pyridin-4-yl-2H-pyrazol-3-yl)-phenoxymethyl]-quinoline (0.5 g,
1.33 mmol) which was synthesized following the method described in J.
Med. Chem. 2009, 52 (16), 5188-5196, in DMF (10 mL) were added methyl
bromoacetate (0.15 mL, 1.60 mmol) and cesium carbonate (1.30 g, 3.99
mmol). The mixture was stirred at room temperature for 3 h. Then it was
quenched with water and extracted with EtOAc. The organic solvents were
separated, dried over sodium sulphate and evaporated to dryness in vacuo.
The crude residue was purified by column chromatography (silicagel;
EtOAc/methanol 100/0 to 95/5). The desired fractions were collected and
evaporated in vacuo to yield a mixture of the two regioisomers I-13 and
I-13' that was used for the next reaction without further purification
(0.47 g, 47%). C27H22N4O3.

[0101] The mixture of intermediate compounds I-13 and 1-13' (0.47 g, 1.043
mmol) was dissolved in ethanol (20 mL) and subjected to hydrogenation in
a H-Cube® system using Pd/C 5% as catalyst (full hydrogen mode, 1.5
mL/min) at 70° C. and atmospheric pressure. The solvent was
evaporated to dryness in vacuo to give a yellow oil that was made solid
by treatment with DCM. The solid was washed with more DCM yielding a
mixture of the two intermediates I-14 and 1-14' that was used for the
next reaction without further purification (0.235 g, 73%).
C17H15N3O3. LCMS: Rt 0.95 (major regioisomer), Rt
1.00 (minor regioisomer), m/z 310 [M+H].sup.+ (method 8).

[0103] A mixture of the two regioisomers I-14 and I-14' (0.235 g, 0.760
mmol), 3,5-dimethyl-2-hydroxymethylpyridine (0.156 g, 1.14 mmol),
diisopropyl azodicarboxylate (0.262 g, 1.14 mmol) and triphenylphosphine
(0.299 g, 1.14 mmol) in THF (6 mL) was heated in a microwave oven at
120° C. for 20 min. After this time the mixture was quenched with
a saturated aqueous solution of sodium carbonate, extracted with EtOAc,
dried over sodium sulphate and the solvent was evaporated to dryness in
vacuo. The crude residue was purified by column chromatography
(silicagel; EtOAc/methanol 100/0 to 95/5). The desired fractions were
collected and the solvent was evaporated in vacuo to yield a mixture of
the two intermediate compounds I-15 and I-15' that was used for the next
reaction without further purification (0.18 g, 55%).
C25H24N4O3.

[0105] To a stirred solution of the mixture of intermediates I-15 and
I-15' (0.18 g, 0.420 mmol) in a mixture of DCM (4 mL) and methanol (1 mL)
was added sodium borohydride (0.079 g, 2.10 mmol). The reaction mixture
was stirred at room temperature for 1 h. The mixture was then quenched
with water, extracted with more DCM, the organic solvent was dried over
sodium sulphate and evaporated to dryness.

[0106] The crude residue was purified by column chromatography (silicagel;
EtOAc/methanol 100/0 to 90/10). The desired fractions were collected and
evaporated in vacuo to yield intermediate compound 1-16 (0.12 g, 61.2%).
The other regioisomer was not isolated from the chromatographic
separation. C24H24N4O2. LCMS: Rt 2.53, m/z 401
[M+H].sup.+ (method 6).

[0108] A mixture of the two regioisomers I-14 and I-14' (0.17 g, 0.550
mmol), (5-methoxy-pyridin-2-yl) methanol (0.115 g, 0.825 mmol) as
described in Bioorg. Med. Chem. 2005, 13, 6763-6770, diisopropyl
azodicarboxylate (0.190 g, 0.825 mmol) and triphenylphosphine (0.216 g,
0.825 mmol) in THF (4 mL) was heated in a microwave oven at 120°
C. for 20 min. After this time the solvent was evaporated to dryness in
vacuo and the crude residue was purified by column chromatography
(silicagel; EtOAc/methanol 100/0 to 95/5). The desired fractions were
collected and the solvent was evaporated in vacuo to yield a mixture of
the two intermediate compounds I-17 and I-17' that was used for the next
reaction without further purification (0.18 g, 76%).
C24H22N4O4.

[0110] To a stirred solution of the mixture of intermediates I-17 and
I-17' (0.18 g, 0.418 mmol) in a mixture of DCM (4 mL) and methanol (1 mL)
was added sodium borohydride (0.079 g, 2.09 mmol). The reaction mixture
was stirred at room temperature for 3 h. The mixture was then quenched
with water, extracted with more DCM, the organic solvent was dried over
sodium sulphate and evaporated to dryness in vacuo. The crude residue was
purified by column chromatography (silicagel; EtOAc/methanol from 100/0
to 90/10). The desired fractions were collected and evaporated in vacuo
to yield intermediate compound 1-18 (0.09 g, 53.5%). The other
regioisomer was not isolated from the chromatographic separation.
C23H22N4O3.

[0113] To a solution of intermediate I-16 (5 mg, 0.012 mmol) in DCM (1 mL)
was added pyridine (11 μL) and this solution was stirred at 0°
C. Methanesulfonic anhydride (16.5 mg, 0.095 mmol)) was then added and
stirring was continued for 4 hours at 0° C. after which the
solvent was evaporated by flushing with nitrogen. The crude mixture was
redissolved in methanol (0.5 mL), diluted with water (4.5 mL) and passed
through a C18 SepPak® cartridge (Waters, Milford, Mass., USA)
that was preconditioned with methanol (3 mL) and milliQ® water (6
mL). The cartridge was then rinsed three times with an additional volume
of water (2 mL) to remove the unreacted methanesulfonic anhydride as much
as possible. The product was eluted from the cartridge using acetonitrile
(3 mL) and the solvents were evaporated under reduced pressure. Prior to
evaporation of the solvents, HPLC analysis was performed to examine the
conversion of the hydroxyl-precursor I-16 into its O-mesyl derivative
I-19. This HPLC analysis was done on an analytical XTerra® RP C18
column (Waters), which was eluted with gradient mixtures of water and
acetonitrile (0 min. 95:5 v/v, 25 min: 10:90 v/v, 30 min: 10:90 v/v,
linear gradient) at a flow rate of 1 mL/min. The analysis showed that the
average conversion rate was 98% (n=9). Residual water was removed by
azeotropic distillation with acetonitrile and the mixture was dried
overnight in the vacuum oven.

[0114] On the day of radiolabelling experiments, which is usually next
day, this reaction product was dissolved in anhydrous DMF (1.5 mL) and
used (0.3 mL) for direct nucleophilic radiofluorination.
C25H26N4O4S.

[0116] To a solution of intermediate 1-18 (5 mg, 0.012 mmol) in DCM (1 mL)
was added pyridine (11 μL) and this solution was stirred at 0°
C. Methanesulfonic anhydride (16.5 mg, 0.095 mmol) was then added and
stirring was continued for 4 hours at 0° C. after which the
solvent was evaporated by flushing with nitrogen. The crude mixture was
redissolved in methanol (0.5 mL), diluted with water (4.5 mL) and passed
through a C18 SepPak® cartridge (Waters, Milford, Mass., USA)
that was preconditioned with methanol (3 mL) and milliQ® water (6
mL). The cartridge was then rinsed three times with an additional volume
of water (2 mL) to remove the unreacted methanesulfonic anhydride as much
as possible. The product was eluted from the cartridge using acetonitrile
(3 mL) and the solvents were evaporated under reduced pressure. Prior to
evaporation of the solvents, HPLC analysis was performed to examine the
conversion of the hydroxyl-precursor I-18 into its O-mesyl derivative
I-20. This HPLC analysis was done on an analytical XTerra® RP C18
column (Waters), which was eluted with gradient mixtures of water and
acetonitrile (0 min: 95:5 v/v, 25 min: 10:90 v/v, 30 min: 10:90 v/v,
linear gradient) at a flow rate of 1 mL/min. The analysis showed that the
average conversion rate was 98% (n=9). Residual water was removed by
azeotropic distillation with acetonitrile and the mixture was dried
overnight in the vacuum oven.

[0117] On the day of radiolabelling experiments, which is usually next
day, this reaction product was dissolved in anhydrous DMF (1.5 mL) and
used (0.3 mL) for direct nucleophilic radio fluorination.
C24H24N4O5S.

[0120] A mixture of intermediate compound 1-5 (0.30 g, 0.876 mmol),
1-bromo-2-fluoroethane (0.083 mL, 1.051 mmol) and cesium carbonate (0.86
g, 2.63 mmol) in DMF (5 mL) was heated in a microwave oven at 150°
C. for 10 min. After this time the reaction mixture was quenched with
water and extracted with EtOAc. The organic layer was dried over sodium
sulphate and the solvent was evaporated to dryness in vacuo.

[0121] The crude residue was purified by column chromatography (silicagel;
EtOAc/methanol 100/0 to 90/10) to give a mixture of the two isomers. This
mixture was further subjected to another column chromatography
purification (silicagel; acetonitrile/methanol 100/0 to 95/5). The
desired fractions were collected and evaporated in vacuo to yield the
desired compound B-1 as an oil (0.15 g, 44.1%). Treatment of this oily
compound with a solution of hydrogen chloride in isopropyl alcohol,
followed by crystallization from diethyl ether/DCM afforded the
hydrochloric acid salt of compound B-1 as a yellow solid.
C23H21FN4O.2HC1. LCMS: Rt 3.82, m/z 389 [M+H].sup.+
(method 3).

[0123] The corresponding regioisomer
2-{4-[2-(2-fluoroethyl)-4-pyridin-4-yl-2H-pyrazol-3-yl]-phenoxymethyl}-6--
methyl-pyridine (B-1') was also isolated from the chromatographic
separation with 70% purity (0.15 g, 30.8%) as an oil.
C23H21FN4O.

Radiosynthesis: Production of [18F]Fluoroethyl Bromide and
[18F]B-1

[0124] [18F]fluoride ([18F]F-) was produced by an
[18O(p,n)18F] reaction by irradiation of 1.95 mL of 97%
enriched [18O]H2O (Rotem HYOX18, Rotem Industries, Beer Sheva,
Israel) in a niobium target using 18 MeV protons from a Cyclone 18/9
cyclotron (Ion Beam Applications, Louvain-la-Neuve, Belgium). After
irradiation for about 60 min, the resultant [18F]F-was
separated from [18O]H2O using a SepPak® Light Accell plus
QMA anion exchange cartridge (Waters), which was preconditioned by
successive treatments with 0.5 M K2CO3 solution (10 mL) and
water (2×10 mL). The [18F]F- was then eluted from the
cartridge into a conical reaction vial (1 mL) using a solution containing
potassium carbonate (2.47 mg) and Kryptofix® 222 (27.92 mg) dissolved
in H2O/CH3CN (0.75 mL; 5:95 v/v). The solvents were evaporated
at 110° C. by applying conventional heating for 2 min. After
evaporation of the solvent, [18F]F- was further dried by
azeotropic distillation of traces of water using acetonitrile (1 mL) at a
temperature of 110° C. until complete dryness.

[0125] A solution of 2-bromoethyl triflate (5 μL, IsoSciences,
Pennsylvania, USA) in o-dichlorobenzene (0.7 mL) was added to the vial
containing [18F]F-. The resulting [18F]FEtBr was then
distilled at 120° C. with a helium flow (3-4 mL/min) and bubbled
into a second reaction vial containing the precursor I-5 (0.2 mg) and a
small amount (1-3 mg) of Cs2CO3 in anhydrous DMF (0.2 mL).
After distillation of a sufficient amount of radioactivity into the
precursor solution, the reaction vial was closed and heated at 90°
C. for 15 min. After the reaction, the crude mixture was diluted with 1.6
mL of water and injected onto the HPLC system consisting of a
semi-preparative XBridge® column (C18, 5 μm, 4.6 mm×150
mm; Waters) that was eluted with a mixture of 0.05 M sodium acetate
buffer pH 5.5 and EtOH (70:30 v/v) at a flow rate of 1 mL/min. UV
detection of the HPLC eluate was performed at 254 nm. The radiolabelled
product [18F]B-1 was collected after about 37 min. (The undesired
isomer elutes after about 45 min). On average 50 mCi (n=2) of purified
[18F]B-1 was collected in 1.5-2 mL volume (mobile phase). The
collected peak corresponding to the [18F]B-1 was then diluted with
normal saline (Mini Plasco®, Braun, Melsungen, Germany) to reduce the
ethanol concentration to <5% and sterile filtered through a 0.22 μm
membrane filter (Millex®-GV, Millipore, Ireland). The purity of the
radiotracer was analyzed using an analytical HPLC system consisting of an
XBridge® column (C18, 3.5 μm, 3 mm×100 mm; Waters)
eluted with a mixture of 0.05 M sodium acetate buffer pH 5.5 and
acetonitrile (70:30 v/v) at a flow rate of 0.8 mL/min (Rt=7.5 min).
[18F]B-1 was synthesized in 57% radiochemical yield (relative to
[18F]FEtBr starting radioactivity n=2). The radiochemical purity as
examined using the above described analytical HPLC system was >99%.

[0129] The corresponding regioisomer
2-{4-[2-(3-fluoropropyl)-4-pyridin-4-yl-2H-pyrazol-3-yl]-phenoxymethyl}-6-
-methyl-pyridine (B-2') was also isolated from the chromatographic
separation and converted into its corresponding hydrochloric acid salt
(0.07 g, 18.2%) as a white powder. C24H23FN4O.

[0131] A mixture of intermediate compound 1-7 (0.20 g, 0.706 mmol),
3,5-dimethyl-2-hydroxymethylpyridine (0.126 g, 0.918 mmol), diisopropyl
azodicarboxylate (0.211 g, 0.918 mmol) and triphenylphosphine (0.241 g,
0.918 mmol) in THF (4 mL) was heated in a microwave oven at 120°
C. for 15 min. After this time the mixture was quenched with a saturated
aqueous solution of sodium carbonate and extracted with DCM. The organic
solvent was dried over sodium sulphate and evaporated to dryness in
vacuo.

[0132] The crude residue was purified by column chromatography (silicagel;
EtOAc/methanol 100/0 to 95/5). The desired fractions were collected and
the solvent was evaporated in vacuo to yield compound B-3 as a colourless
oil. The residue was dissolved in methanol (2 mL) and a solution of
succinic acid (0.073 g, 0.619 mmol) in methanol (2 mL) was slowly added.
The solvent was evaporated to dryness and the solid residue was washed
several times with diethyl ether, yielding the succinic acid salt of
final compound B-3 (0.295 g, 80.3%) as a white solid.
C24H23FN4O.C4H6O4. LCMS: Rt 3.08, m/z 403
[M +H].sup.+(method 7).

[0134] [18F]fluoride ([18F]F-) was produced in a similar
way as described above for the radiosynthesis of [18F]FEtBr with the
modification that the [18F]F- was eluted from the cartridge
using a solution of 0.45 mL of the Kryptofix® 222/K2CO3
solution and 0.3 mL of acetonitrile.

[0135] The radiolabeling precursor I-19 (˜0.6 mg in 0.3 mL DMF) was
added to the dried [18F]F-/K2CO3/Kryptofix® 222
complex and the nucleophilic substitution reaction was carried out by
conventional heating at 90° C. for 15 min. After the reaction, the
crude mixture was diluted with 1.4 mL of water and injected onto the HPLC
system consisting of a semi-preparative XBridge® column (C18, 5
μm, 4.6 mm×150 mm; Waters) that was eluted with a mixture of
0.05 M sodium acetate buffer pH 5.5 and EtOH (65:35 v/v) at a flow rate
of 1 mL/min. UV detection of the HPLC eluate was performed at 254 nm. The
radiolabelled product [18F]B-3 was collected after about 26 min. On
average 100 mCi (n=7, min 60 mCi, max 180 mCi) of purified [18F]B-3
was collected in 1.5-2 mL volume (mobile phase). The collected peak
corresponding to the [18F]B-3 was then diluted with normal saline
(Mini Plasco®, Braun, Melsungen, Germany) to reduce the ethanol
concentration to <5% and sterile filtered through a 0.22 μm
membrane filter (Millex®-GV, Millipore, Ireland). The purity of the
radiotracer was analyzed using an analytical HPLC system consisting of an
XBridge® column (C18, 3.5 nm, 3 mm×100 mm; Waters) eluted
with a mixture of 0.05 M sodium acetate buffer pH 5.5 and acetonitrile
(65:35 v/v) at a flow rate of 0.8 mL/min (Rt=5.6 min) UV detection of the
HPLC eluate was performed at 254 nm. [18F]B-3 was synthesized in 16%
radiochemical yield (relative to starting radioactivity
[18F]F-, n=7). The radiochemical purity as examined using the
above described analytical HPLC system was >98%. The average specific
radioactivity of the tracer as examined using the above described
analytical HPLC system was found to be 176 GBq/nmol (4764 Ci/mmol, n=7)
at the end of synthesis (EOS).

[0139] [18F]fluoride ([18F]F-) was produced exactly in the
same way as described above for the radiosynthesis of compound
[18F]B-3.

[0140] The radiolabeling precursor I-20 (˜0.6 mg in 0.3 mL DMF) was
added to the dried [18F]F-/K2CO3/Kryptofix® 222
complex and the nucleophilic substitution reaction was carried out by
conventional heating at 90° C. for 10 min. After the reaction, the
crude mixture was diluted with 1.4 mL of water and injected onto the HPLC
system consisting of a semi-preparative XBridge® column (C18, 5
μtm, 4.6 mm×150 mm; Waters) that was eluted with a mixture of
0.05 M sodium acetate buffer pH 5.5 and EtOH (70:30 v/v) at a flow rate
of 1 mL/min. UV detection of the HPLC eluate was performed at 254 nm. The
radiolabelled product [18F]B-4 was collected after about 35 min.
Typically, about 90 mCi of purified [18F]B-4 was collected in 1.5-2
mL volume (mobile phase). The collected peak corresponding to the
[18F]B-4 was then diluted with normal saline (Mini Plasco®,
Braun, Melsungen, Germany) to reduce the ethanol concentration to <5%
and sterile filtered through a 0.22 lam membrane filter
(Millex®-GV, Millipore, Ireland). The purity of the radiotracer was
analyzed using an analytical HPLC system consisting of an XBridge®
column (C18, 3.5 μm, 3 mm×100 mm; Waters) eluted with a
mixture of 0.05 M sodium acetate buffer pH 5.5 and acetonitrile (70:30
v/v) at a flow rate of 0.8 mL/min (Rt=7.2 min) UV detection of the HPLC
eluate was performed at 254 nm. [18F]B-4 was synthesized in 15%
radiochemical yield (relative to starting radioactivity
[18F]F-, n=2). The radiochemical purity as examined using the
above described analytical HPLC system was >99%. The average specific
radioactivity of the tracer as examined using the above described
analytical HPLC system was found to be 141 GBq/μmol (3800 Ci/mmol,
n=2) at the EOS.

[0145] A mixture of intermediate compound I-7 (0.30 g, 1.06 mmol),
(6-bromo-pyridin-2-yl)methanol (0.30 g, 1.59 mmol), diisopropyl
azodicarboxylate (0.315 mL, 1.59 mmol) and triphenylphosphine (0.417 g,
1.59 mmol) in THF (5 mL) was heated in a microwave oven at 100° C.
for 30 min. After this time the mixture was quenched with a saturated
aqueous solution of sodium carbonate and extracted with DCM. The organic
solvent was dried over sodium sulphate and evaporated to dryness in
vacuo. The crude residue was purified by column chromatography
(silicagel; first EtOAc/heptane 70/30 and then diethyl ether/DCM 70/30).
The desired fractions were collected and the solvent was evaporated in
vacuo to yield compound B-6 (0.30 g, 53.1%) as a colourless oil. An
amount of compound B-6 (0.08 g) was converted into the corresponding
succinic acid salt in a similar way as it is described above for final
compound B-3, yielding final compound B-6 as a white solid.
C22H18BrFN4O.0.75C4H6O4. LCMS: Rt 3.22, m/z
453 [M+H].sup.+ (method 7).

[0148] Compound B-6 (0.45 g, 0.725 mmol) was dissolved in DMF (3 mL) and
then (fluorosulfonyl)difluoroacetic acid methyl ester (0.464 mL, 3.62
mmol) and cuprous iodide (0.69 g, 3.62 mmol) were added to the solution.
The reaction mixture was heated at 120° C. in a sealed tube for 2
h. The reaction mixture was quenched with aqueous 1 M sodium hydroxide
and extracted with DCM. The organic solvent was dried over sodium
sulphate and evaporated to dryness in vacuo. The residue was purified by
column chromatography (silicagel; EtOAc/heptane 70/30 to 100/0). The
desired fractions were collected and the solvent was evaporated in vacuo
to give the desired compound as a yellow oil, which contained
triphenylphosphine oxide as the main impurity. The crude compound was
further purified by preparative HPLC (C18 XBridge 30×100; aq.
ammonium carbonate pH 9/acetonitrile gradient from 80/20 to 0/100)
affording B-7 as a colourless oil. The compound was made solid by
addition of diethyl ether/heptane and finally was recrystallized from
diisopropyl ether yielding final compound B-7 (0.023 g, 7.2%) as a white
solid. C23F118F4N4O. LCMS: Rt 3.73, m/z 443
[M+H].sup.+ (method 4).

[0151] A mixture of compound B-6 (0.14 g, 0.263 mmol), cyclopropyl-boronic
acid (0.029 g, 0.341 mmol) and palladium (0) tetrakis(triphenylphosphine)
(0.015 g, 0.013 mmol) in a mixture of aq. sodium carbonate/dioxane 1:1 (5
mL) was heated in a microwave oven at 130° C. for 15 min. After
cooling to room temperature, the crude mixture was diluted with water and
extracted with DCM. The organic solvent was dried over sodium sulphate
and evaporated to dryness in vacuo. The residue was purified by column
chromatography (silicagel; EtOAc), the desired fractions were collected
and the solvent was evaporated in vacuo to give the desired compound as a
colourless oil, which contained triphenylphosphine oxide as the main
impurity. The compound was further purified by preparative HPLC (C18
XBridge 19×100; aq. ammonium carbonate pH 9/acetonitrile gradient
from 80/20 to 0/100) affording B-8 as a colourless oil. The residue was
dissolved in methanol (2 mL) and a solution of succinic acid (0.017 g,
0.144 mmol) in methanol (1 mL) was slowly added. The solvent was
evaporated to dryness and the residue was treated with DCM/diisopropyl
ether, yielding the succinic acid salt of final compound B-8 (0.076 g,
54.4%) as a white solid.

[0156] Following the procedure for the preparation of compound B-5 but
substituting (5-methoxypyridin-2-yl)methanol for
(3-methoxypyridin-2-yl)methanol provided final compound B-9 (58%) as a
white solid that was further crystallized from diisopropyl ether.
C23H19F3N4O2. LCMS: Rt 3.01, m/z 441 [M+H].sup.+
(method 7)).

[0158] Following the procedure for the preparation of compound B-5 but
substituting (5-methoxypyridin-2-yl)methanol for
[3-(2-fluoroethoxy)-pyridin-2-yl]-methanol provided final compound B-10
that was converted into the corresponding succinic acid salt, in a
similar way as it is described above for final compound B-3, which was
crystallized from diethyl ether yielding the succinic acid salt of final
compound B-10 (33.8%) as a white solid.
C24H20F4N4O2.C4H6O4. LCMS: Rt
2.96, m/z 473 [M+H].sup.+ (method 7).

[0162] Following the procedure for the preparation of compound B-5, but
substituting (5-methoxypyridin-2-yl)methanol for
(3-methoxypyridin-2-yl)methanol and 1-10 for intermediate compound 1-8,
provided final compound B-12 (40%) as a white solid.

[0165] Following the procedure for the preparation of compound B-5, but
substituting (5-methoxypyridin-2-yl)methanol for
[3-(2-fluoroethoxy)-pyridin-2-yl]-methanol and 1-10 for intermediate
compound 1-8, provided final compound B-13 that was converted into the
corresponding succinic acid salt, in a similar way as it is described
above for final compound B-3, which was crystallized from diethyl
ether/diiisopropyl ether yielding the succinic acid salt of final
compound B-13 (39.2%) as a white solid.

[0168] Following the procedure for the preparation of compound B-5, but
substituting (5-methoxypyridin-2-yl)methanol for
(5-methoxypyridin-2-yl)methanol and 1-10 for intermediate compound 1-8,
provided final compound B-14 as a colourless oil, that was converted into
the corresponding succinic acid salt, in a similar way as it is described
above for final compound B-3, which was crystallized from diethyl ether
yielding the succinic acid salt of final compound B-14 (25.6%) as a white
solid.

[0171] Following the procedure for the preparation of compound B-5, but
substituting (5-methoxypyridin-2-yl)methanol for
[5-(2-fluoroethoxy)-pyridin-2-yl]-methanol and 1-10 for intermediate
compound 1-8, provided compound B-15 as a colourless oil that solidified
on standing. Finally the compound was washed with diisopropyl ether to
yield final compound B-15 (35.8%) as a white solid.
C25H24F2N4O2. LCMS: Rt 3.69, m/z 451 [M+H].sup.+
(method 5).

[0173] Following the procedure for the preparation of compound B-4 but
substituting (5-methoxypyridin-2-yl)methanol for
(3-methoxypyridin-2-yl)methanol provided compound B-16 that was converted
into the corresponding succinic acid salt, in a similar way as it is
described above for final compound B-3, which was crystallized from
diethyl ether yielding the succinic acid salt of final compound B-16
(32.5%) as a white solid.
C23H21FN4O2.C4H6O4. LCMS: Rt 3.52, m/z
405 [M+H].sup.+ (method 2).

[0175] Following the procedure for the preparation of compound B-4 but
substituting (5-methoxypyridin-2-yl)methanol for
[3-(2-fluoroethoxy)-pyridin-2-yl]-methanol provided compound B-17 that
was converted into the corresponding succinic acid salt, in a similar way
as it is described above for final compound B-3, which was crystallized
from diethyl ether yielding the succinic acid salt of final compound B-17
(78.3%) as a white solid.
C24H22F2N4O2.C4H6O4. LCMS: Rt
2.52, m/z 437 [M +Fl].sup.+ (method 7).

[0177] Following the procedure for the preparation of compound B-6 but
substituting (6-bromopyridin-2-yl)methanol for
(3-fluoropyridin-2-yl)methanol provided compound B-18 that was converted
into the corresponding succinic acid salt, in a similar way as it is
described above for final compound B-3, which was crystallized from
diethyl ether yielding the succinic acid salt of final compound B-18
(23.9%) as a white solid.

[0182] Following the procedure for the preparation of compound B-6 but
substituting (6-bromopyridin-2-yl)methanol for
(5-bromopyridin-2-yl)methanol provided compound B-20 (15.6%) as a white
solid after treatment with diethyl ether. C22H18BrFN4O.

[0191] Following the procedure for the preparation of compound B-4, but
substituting (5-methoxypyridin-2-yl)methanol for
(5-cyclopropyl-pyridin-2-yl)methanol provided compound B-23 as a
colourless oil that solidified on standing. Finally the compound was
washed with diisopropyl ether to yield final compound B-23 (45.6%) as a
white solid. C25H23FN4O. LCMS: Rt 3.24, m/z 415
[M+H].sup.+(method 7).

[0193] Following the procedure for the preparation of compound B-4 but
substituting (5-methoxypyridin-2-yl)methanol for
(6-methoxypyridin-2-yl)methanol provided compound B-24 that was converted
into the corresponding succinic acid salt, in a similar way as it is
described above for final compound B-3, which was crystallized from
diethyl ether yielding the succinic acid salt of final compound B-24
(25.8%) as a white solid.
C23H21FN4O2.C4H6O4. LCMS: Rt 4.16, m/z
405 [M+H].sup.+ (method 2).

[0195] Following the procedure for the preparation of compound B-4 but
substituting (5-methoxypyridin-2-yl)methanol for
(6-ethoxypyridin-2-yl)methanol provided compound

[0196] B-25 that was converted into the corresponding succinic acid salt,
in a similar way as it is described above for final compound B-3, which
was crystallized from diethyl ether yielding the succinic acid salt of
final compound B-25 (39.6%) as a white solid.
C24H23FN4O2.C4H6O4. LCMS: Rt 3.56, m/z
419 [M+H].sup.+ (method 7).

[0198] Following the procedure for the preparation of compound B-6 but
substituting (6-bromopyridin-2-yl)methanol for
6-hydroxymethyl-2-cyanopyridine provided compound B-26 (40.8%) as a white
solid after treatment with diethyl ether.

[0203] Following the procedure for the preparation of compound B-3 but
substituting 3,5-dimethyl-2-hydroxymethylpyridine for
(3,4,5-trimethylpyridin-2-yl)methanol provided final compound B-28
(56.8%) in its succinic acid salt form as a white solid.

[0206] Rat recombinant PDE10A (rPDE10A) was expressed in Sf9 cells using a
recombinant rPDE10A baculovirus construct. Cells were harvested after 48
h of infection and the rPDE10A protein was purified by metal chelate
chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and
diluted in 100% DMSO to a concentration 100 fold of the final
concentration in the assay. Compound dilutions (0.4 μl) were added in
384 well μlates to 20 μl of incubation buffer (50 mM Tris pH 7.8,
8.3 mM MgCl2, 1.7 mM EGTA). 10 μl of rPDE10A enzyme in incubation
buffer was added and the reaction was started by addition of 10 μl
substrate to a final concentration of 60 nM cAMP and 0.008 μCi
3H-cAMP. The reaction was incubated for 60 minutes at room temperature.
After incubation, the reaction was stopped with 20 μl of 17.8 mg/ml
PDE SPA beads. After sedimentation of the beads during 30 minutes, the
radioactivity was measured in a Perkin Elmer Topcount scintillation
counter and results were expressed as cpm. For blanc values the enzyme
was omitted from the reaction and replaced by incubation buffer. Control
values were obtained by addition of a final concentration of 1% DMSO
instead of compound. A best fit curve was fitted by a minimum sum of
squares method to the plot of % of control value substracted with blanc
value versus compound concentration and a pIC50 value was derived from
this curve.

[0207] Values are peak values, and are obtained with experimental
uncertainties that are commonly associated with this analytical method.

[0208] For a number of compounds, noted as "DSC" in the above table,
melting points were determined with a DSC823e (Mettler-Toledo). Melting
points were measured with a temperature gradient of 30° C./minute.
Maximum temperature was 400° C.

[0209] For a number of compounds, melting points were determined in open
capillary tubes on a Mettler FP62 apparatus. Melting points were measured
with a temperature gradient of 10° C./minute. Maximum temperature
was 300° C. The melting point was read from a digital display.

Nuclear Magnetic Resonance (NMR)

[0210]1H NMR spectra were recorded either on a Bruker DPX-400 or on
a Bruker AV-500 spectrometer with standard pulse sequences, operating at
400 MHz and 500 MHz respectively. Chemical shifts (δ) are reported
in parts per million (ppm) downfield from tetramethylsilane (TMS), which
was used as internal standard.

LCMS-Methods:

[0211] For LCMS-characterization of the compounds of the present
invention, the following methods were used.

General Procedure for HP 1100-MS Instruments (TOF or SOD)

[0212] The HPLC measurement was performed using an HP 1100 (Agilent
Technologies) system comprising a pump (quaternary or binary) with
degasser, an autosampler, a column oven, a diode-array detector (DAD) and
a column as specified in the respective methods. The MS detector was
configured with either an electrospray ionization source or an ESCI dual
ionization source (electrospray combined with atmospheric pressure
chemical ionization). Nitrogen was used as the nebulizer gas. The source
temperature was maintained either at 140° C. or 100° C.
Data acquisition was performed either with MassLynx-Openlynx software or
Chemsation-Agilent Data Browser software.

General Procedure for Acquity-SOD Instrument

[0213] The UPLC (Ultra Performance Liquid Chromatography) measurement was
performed using an Acquity UPLC (Waters) system comprising a sampler
organizer, a binary pump with degasser, a four column's oven, a
diode-array detector (DAD) and a column as specified in the respective
methods. The MS detector was configured with an ESCI dual ionization
source (electrospray combined with atmospheric pressure chemical
ionization). Nitrogen was used as the nebulizer gas. The source
temperature was maintained at 140° C. Data acquisition was
performed with MassLynx-Openlynx software.

[0214] MS Procedure for LC Methods 1 and 2: High-resolution mass spectra
(Time of Flight, TOF detector) were acquired only in positive ionization
mode or in positive/negative modes by scanning from 100 to 750 umas. The
capillary needle voltage was 2.5 kV for positive mode 2.9 Kv for negative
ionization mode. The cone voltage was 20 V for both positive and negative
ionization modes. Leucine-Enkephaline was the standard substance used for
the lock mass calibration.

[0216] In addition to the general procedure: Reversed phase HPLC was
carried out on an XDB-C18 cartridge (1.8 μm, 2.1×30 mm) from
Agilent, at 60° C. with a flow rate of 1 ml/min, at 60° C.
The gradient conditions used are: 90% A (0.5 g/l ammonium acetate
solution), 5% B (acetonitrile), 5% C (methanol) to 50% B and 50% C, then
to 100% B and equilibrated to initial conditions up to 9.0 minutes run.
Injection volume 2 μl.

Method 2

[0217] In addition to the general procedure: Reversed phase HPLC was
carried out on a Sunfire-C18 column (2.5 μm, 2.1×30 mm) from
Waters, with a flow rate of 1.0 ml/min, at 60° C. The gradient
conditions used are: 95% A (0.5 g/l ammonium acetate solution +5% of
acetonitrile), 5% B (acetonitrile or acetonitrile/methanol 1/1), to 100%
B and equilibrated to initial conditions up to 9 or 7 minutes run.
Injection volume 2 μl.

Method 3

[0218] In addition to the general procedure: Reversed phase HPLC was
carried out on a XDB-C18 cartridge (1.8 μm, 2.1×30 mm) from
Agilent, with a flow rate of 0.8 ml/min, at 60° C. The gradient
conditions used are: 90% A (0.5 g/l ammonium acetate solution), 10% B
(mixture of Acetonitrile/ Methanol, 1/1), to 100% B and equilibrated to
initial conditions up to 9.0 minutes run. Injection volume 2 μl.

Method 4

[0219] In addition to the general procedure: Reversed phase HPLC was
carried out on a Sunfire-C18 column (2.5 μm, 2.1×30 mm) from
Waters, with a flow rate of 1.0 ml/min, at 60° C. The gradient
conditions used are: 95% A (0.5 g/l ammonium acetate solution +5%
acetonitrile), 5% B (mixture of acetonitrile/methanol, 1/1), to 100% B
and equilibrated to initial conditions up to 7 minutes run. Injection
volume 2 μl.

Method 5

[0220] In addition to the general procedure: Reversed phase HPLC was
carried out on a XBridge-C18 column (2.5 μm, 2.1×30 mm) from
Waters, with a flow rate of 1.0 ml/min, at 60° C. The gradient
conditions used are: 95% A (0.5 g/l ammonium acetate solution +5%
acetonitrile), 5% B (mixture of acetonitrile/methanol, 1/1), to 100% B
and equilibrated to initial conditions up to 9.0 minutes run. Injection
volume 2 μl.

Method 6

[0221] In addition to the general procedure: Reversed phase HPLC was
carried out on an Eclipse Plus-C18 column (3.5 μm, 2.1×30 mm)
from Agilent, with a flow rate of 1.0 ml/min, at 60° C. The
gradient conditions used are: 95% A (0.5 g/l ammonium acetate solution
+5% acetonitrile), 5% B (acetonitrile or mixture of
acetonitrile/methanol, 1/1), to 100% B and equilibrated to initial
conditions up to 7 minutes run. Injection volume 2 μl.

Method 7

[0222] In addition to the general procedure: Reversed phase UPLC was
carried out on a BEH-C18 column (1.7 μm, 2.1×50 mm) from Waters,
with a flow rate of 0.8 ml/min, at 60° C. The gradient conditions
used are: 95% A (0.5 g/l ammonium acetate solution +5% acetonitrile), 5%
B (mixture of acetonitrile/methanol, 1/1), to 20% A, 80% B, then to 100%
B and equilibrated to initial conditions up to 7 or 5 minutes run.
Injection volume 0.5 μl.

Method 8

[0223] In addition to the general procedure: Reversed phase UPLC was
carried out on a BEH-C18 column (1.7 μm, 2.1×50 mm) from Waters,
with a flow rate of 1.0 ml/min, at 50° C. The gradient conditions
used are: 95% A (0.5 g/l ammonium acetate solution +5% acetonitrile), 5%
B (acetonitrile), to 40% A, 60% B, then to 5% A, 95% B and equilibrated
to initial conditions up to 5 minutes run. Injection volume 0.5 μl.

II. Biodistribution studies:

General Method

[0224] Biodistribution studies were carried out in healthy male Wister
rats (body weight 200-500 g) at 2 min, 30 min and 60 min post injection
(p.i.) (n=3/time point). For |18F|B-4 only the 2 min and 30 min time
points were studied. Rats were injected with about 30 μCi of the
tracer via tail vein under anesthesia (2.5% isoflurane in O2 at 1
L/min flow rate) and sacrificed by decapitation at above specified time
points. Blood and major organs were collected in tared tubes and weighed.
The radioactivity in blood, organs and other body parts was measured
using an automated gamma counter. The distribution of radioactivity in
different parts of the body at different time points p.i. of the tracer
was calculated and expressed as percentage of injected dose (% ID), and
as percentage of injected dose per gram tissue (% ID/g) for the selected
organs. % ID is calculated as cpm in organ/total cpm recovered. For
calculation of total radioactivity in blood, blood mass was assumed to be
7% of the body mass.

A. Biodistribution Results for Compound [18F]B-1

[0225] The results of the in vivo distribution study of [18F]B-1 in
male Wistar rats is presented in Tables 1 and 2. Table 1 shows the % ID
values at 2 min, 30 min and 60 min p.i. of the radiotracer. At 2 min p.i.
about 4.0% of the injected dose was present in the blood, and this
cleared to 2.2% by 60 min after injection of the tracer. The total
initial brain uptake of the tracer was 0.59%, with 0.49% of the ID in the
cerebrum and 0.09% in the cerebellum. At 60 min after injection of the
radiotracer, 54% ID was present in the liver and intestines. Because of
its lipophilic character, the urinary excretion of the tracer was minimal
with only ˜2.6% ID present in the urinary system at 60 min p.i.
Increasing accumulation in the stomach was observed (3% ID, 10% ID, 17%
ID at respectively 2, 30 and 60 min p.i.) In view of the large mass of
the carcass, significant amount of the injected dose (˜28% ID) was
present in the carcass at all time points examined. Typically, carcass
constitutes to >90% of the total body weight of the animal. Table 2
shows the % ID/g values for different organs at 2 min, 30 min and 60 min
p.i.

[0226] As kidneys and liver are the excretory organs, they have the
highest % ID/g values with about 1.8% ID/g for kidneys and 3.4% ID/g for
liver at 2 min p.i. The % ID/g values for different regions of brain,
namely striatum, hippocampus, cortex and cerebellum are presented in
Table 2. In order to correct for differences in body weight between
different animals, the % ID/g tissue values were normalized for body
weight. The normalized values are presented in Table 3. For all studied
brain regions there is a significant decrease in radioactivity
concentration from 2 to 30 min (≦0.07% ID/g corrected for body
weight at 30 min p.i.), indicating significant washout of the tracer from
all studied brain regions.

[0227] Table 4 shows the 2 min/30 min and 2 min/60 min ratios of % ID/g
values (normalized for body weight of the animal) for different regions
of the brain. For all brain regions the 2 min/30 min ratios are
≧2.49 indicating that the tracer already started to washout during
this time period. Slowest washout is observed for striatum, the region
with the highest expression of PDE10.

[0228] Table 5 presents the ratios between striatum and other regions of
the brain as well as blood at different time points post injection of
[18F]B-1. Striatum is considered as the PDE10A-rich region and
cerebellum as the reference region. Therefore high striatum-to-cerebellum
ratios are desired in order to have good quality images in vivo. The
maximum striatum-to-cerebellum ratio was about 2, which is rather low for
a good PDE10 imaging tracer.

[0229] The results of these biodistribution studies indicate that there is
no significant retention of [18F]B-1 in the PDE10A-rich region
striatum.

[0231] The results of the in vivo distribution study of [18F]B-3 in
male Wistar rats is presented in Tables 6 and 7. Table 6 shows the % ID
values at 2 min, 30 min and 60 min p.i. of the radiotracer. The total
initial brain uptake of the tracer was rather low: 0.39% of the ID at 2
min p.i., with 0.31% ID in the cerebrum and 0.07% ID in the cerebellum.
At 30 min p.i. 0.035% of ID was present in striatum, which has the
highest expression of PDE10A, and where the radiotracer is expected to
show binding. Clearance from blood circulation was rather slow. At 2 min
p.i. about 4.0% of the injected dose was present in the blood, and this
cleared to 3.3% by 60 min p.i. The tracer was cleared mainly by the
hepatobiliary system as there was in total 50% of ID present in liver and
intestines 60 min after injection of the radiotracer. Because of its
lipophilic character, the urinary excretion of the tracer was minimal
with only ˜3.2% ID present in the urinary system at 60 min p.i.
There is also an unexpected high accumulation in the stomach (2.6% ID,
6.7%ID, 20% ID at respectively 2, 30 and 60 min p.i.). In view of the
large mass of the carcass, significant amount of the injected dose
(˜27% ID) was present in the carcass at all time points examined
Typically, carcass constitutes to >90% of the total body weight of the
animal.

[0233] As kidneys and liver are the excretory organs, they have the
highest % ID/g values with about 1.9% ID/g for kidneys and 5.5% ID/g for
liver at 2 min p.i. The % ID/g values for different regions of brain,
namely striatum, hippocampus, cortex and cerebellum are presented in
Table 7. In order to correct for differences in body weight between
different animals, the % ID/g tissue values were normalized for body
weight. The normalized values are presented in Table 8. At 2 min p.i. the
highest radioactivity concentration in brain is observed in the striatum
region (0.098% ID/g corrected for body weight) and this increases in time
(0.121% ID/g corn at 30 min and 0.178% ID/g corr. at 60 min p.i.). This
accumulation of radioactivity in striatum is consistent with the higher
expression of the PDE10 enzyme in this region. For hippocampus, cortex
and cerebellum, regions with minimal expression of PDE10A, the
concentration at 30 min is decreased compared to the 2 min time point,
indicating washout of the tracer from these brain regions. The slight
increase at 60 min for these brain regions can be due to formation of
radiometabolite(s) that enter the brain.

[0234] Table 9 shows 2 min/30 min and 2 min/60 min ratios of % ID/g values
(normalized for body weight of the animal) for different regions of the
brain. For the 2 min/30 min ratios, the cerebellum has the highest ratio
of 2.71, indicating that the clearance of the tracer is the fastest from
this region, followed by cortex (2.54) and hippocampus (2.14), regions
with minimal expression of PDE10. For the striatum, on the other hand,
the 2 min/60 min ratio (0.55) was lower than the 2 min/30 min ratio
(0.81), indicating accumulation of [18F]3-3 in the PDE10A-rich
striatum.

[0235] Table 10 presents the ratios between striatum and other regions of
the brain as well as blood at different time points post injection of
[18F]B-3. Striatum is considered as the PDE10A-rich region and
cerebellum as the reference region. Therefore high striatum-to-cerebellum
ratios are desired in order to have good quality images in vivo. At 2 min
p.i., the striatum-to-cerebellum ratio was about 1.6 and this ratio
increased to 6.6 by 60 min after injection of the tracer.
Striatum-to-cortex and striatum-to-hippocampus ratios were also
≧6.5 (60 min p.i.), confirming the specific retention of
[18F]B-3 in striatum.

[0236] The results from these biodistribution studies indicate that
although the initial brain uptake was rather low, there is a continuous
accumulation of [18F]B-3 in striatum, while there is a washout in
time from the reference region cerebellum. This suggests specific
retention of [18F]B-3 in the PDE10A-rich region striatum.

C. Biodistribution Results for Compound [18F]B-4

[0237] The results of the in vivo distribution study of [18F]B-4 in
male Wistar rats is presented in Tables 11 and 12. Because of the rather
low initial brain uptake and poor accumulation in striatum, the 60 min
biodistribution analysis was not performed. Table 11 shows the % ID
values at 2 min and 30 min p.i. of the radiotracer. The total initial
brain uptake of the tracer was rather low: 0.40% of the ID at 2 min p.i.,
with 0.32% ID in the cerebrum and 0.07% ID in the cerebellum. At 30 min
p.i. 0.019% of ID was present in striatum, which is rather low since the
striatum has the highest expression of PDE10A, and in this region of the
brain the radiotracer is expected to show binding. The tracer was cleared
mainly via the liver (47% ID at 30 min p.i.) into the intestines (12% ID
at 30 min p.i.) Because of its lipophilic character, the urinary
excretion of the tracer was minimal with only ˜2.5% ID present in
the urinary system at 30 min p.i. There is also an unexpected high
accumulation in the stomach (2.8% ID and 8% ID at respectively 2 and 30
min p.i.). Table 12 shows the % ID/g values for different organs at 2 min
and 30 min p.i.

[0238] As kidneys and liver are the excretory organs, they have the
highest % ID/g values with about 1.3% ID/g for kidneys and 2.3% ID/g for
liver at 2 min p.i. The % ID/g values for different regions of brain,
namely striatum, hippocampus, cortex and cerebellum are presented in
Table 12. In order to correct for differences in body weight between
different animals, the % ID/g tissue values were normalized for body
weight. The normalized values are presented in Table 13. At 2 min p.i.
the highest radioactivity concentration in brain is observed in striatum
(0.226% ID/g corrected for body weight). For hippocampus, cortex and
cerebellum this value was respectively 0.091% ID/g corr., 0.168% ID/g
corr., 0.109% ID/g corr. At 30 min p.i. the concentration in striatum
decreased to 0.125% ID/g corrected for body weight. For the other brain
regions the washout was higher (≦0.040% ID/g corrected for body
weight at 30 min p.i.).

[0239] Table 14 shows 2 min/30 min and 2 min/60 min ratios of % ID/g
values (normalized for body weight) for different regions of the brain.
For the 2 min/30 min ratios, the cortex has the highest ratio of 4.24,
indicating that the clearance of the tracer is fastest from this region,
followed by cerebellum (3.82) and hippocampus (3.08), which do not
express PDE10. For the striatum, the 2 min/30 min ratio was the lowest
(1.80), indicating slower washout of [18F]B-4 from the PDE10-rich
region striatum.

[0240] Table 15 presents the ratios between striatum and other regions of
the brain as well as blood at different time points post injection of
[18F]B-4. Striatum is considered as the PDE10A-rich region and
cerebellum as the reference region. Therefore high striatum-to-cerebellum
ratios are desired in order to have good quality images in vivo. At 2 min
p.i., the striatum-to-cerebellum ratio was about 2.07 and this ratio
increased to 4.38 by 30 min after injection of the tracer.
Striatum-to-cortex and striatum-to-hippocampus ratios were also >3.16,
confirming retention of [18F]B-4 in striatum.

[0241] The metabolic stability of compound [18F]B-3 was studied in
normal rats (n=2) by determination of the relative amounts of parent
tracer and radiometabolites in plasma at 2, 30 and 60 min p.i. of the
tracer. After intravenous (i.v.) administration of about 1.7 mCi
[18F]B-3 via tail vein under anesthesia (2.5% isoflurane in O2
at 1 L/min flow rate), blood was collected via the tail vein at the above
mentioned time points (from the same animal) in lithium heparin
containing tubes (4.5 mL LH PST tubes; BD vacutainer, BD, Franklin Lakes,
USA) and stored on ice to stop the metabolism. Next, the blood was
centrifugated for 10 min at 3000 rpm to separate the plasma. A volume of
about 0.1 mL of plasma sample was isolated and spiked with about 10 μL
of authentic non-radioactive B-3 (1 mg/mL solution) and 10 μL of
intermediate I-7 (1 mg/mL solution, major metabolite according to in
vitro studies) for identification. The plasma was then injected onto an
HPLC system consisting of a Chromolith Performance column (C18, 3
mm×100 mm, Merck) that was eluted with mixtures of 0.05 M NaOAc pH
5.5 (solvent A) and acetonitrile (solvent B). The following method was
used for the analysis: isocratic elution with 100% A for 4 min at a flow
rate of 0.5 mL/min, then linear gradient to 90% B by 14 min at a flow
rate of 1 mL/min, and isocratic elution with a mixture of 10% A and 90% B
at a flow rate of 1 mL/min until 17 min. After passing through an in-line
UV detector (254 nm), the HPLC eluate was collected as 1-mL fractions
(fraction collection each min) using an automatic fraction collector. For
good separation between the intact tracer and the polar radiometabolite
[18F]I-7, fractions were collected each 30 sec starting from 8 min
post HPLC injection until 12 min post HPLC injection. The radioactivity
in all fractions was measured using an automated gamma counter. The peak
corresponding to the intact [18F]B-3 eluted at ˜18 min,
whereas the expected radiometabolite [18F]I-7 eluted from ˜13
to 15 min. An unidentified polar radiometabolite eluting from 3 to 5 min
was also detected in brain. An overview of the results from the plasma
radiometabolite analysis is presented in Table 16.

[0242] The analysis shows that at 2 min post injection of the radiotracer,
about 96% of the recovered radioactivity in plasma was in the form of
intact tracer. At 30 min p.i. 47% of polar radiometabolites were detected
in plasma and this amount increased to 62% at 60 min p.i. The most polar
radiometabolite probably anises from the cleavage and oxidation of the
[18F]fluoro-ethyl chain as this polar metabolite is also detected in
brain (vide infra). The second polar radiometabolite, eluting closely to
the intact tracer, could be identified as [18F]I-7. The
radioactivity corresponding to the (more lipophilic) fractions eluting
after the intact tracer were negligible and this indicates no formation
of lipophilic radiometabolites which, if present, could penetrate the
blood-brain-barrier.

B. Perfused Brain Radiometabolite Analysis at 30/60 Minutes p.i.

[0243] For each studied time point two rats were injected with about 0.6
mCi of [18F]B-3. At 30 min or 60 min after injection of the tracer,
the rats were sacrificed by administering an overdose of Nembutal. When
breathing had stopped, the rats were perfused with saline (Mini
Plasco®, Braun, Melsungen, Germany) until the liver turned pale.
Brain was isolated, cerebrum and cerebellum were separated and
homogenized in 3 mL and 2 mL of acetonitrile, respectively, for about 2
min. A volume of 1 mL of this homogenate was diluted with an equal volume
of water and a part of this homogenate was filtered through a 0.22 μm
filter (Millipore, Bedford, USA). About 0.4 mL of the filtrate was
diluted with 0.1 mL of water and spiked with 10 μL of authentic
non-radioactive B-3 (1 mg/mL solution) and 10 μL of I-7 (1 mg/mL
solution, major metabolite according to in vitro studies) for
identification. The cerebrum/cerebellum homogenate was then injected onto
an HPLC system consisting of an analytical XBridge® column (C18,
5 μM, 3 mm×100 mm, Waters) eluted with a mixture of 0.05 M
sodium acetate buffer pH 5.5 and acetonitrile (70:30 v/v) at a flow rate
of 0.8 mL/min. The HPLC eluate was collected as 1-mL fractions (fraction
collection each minute) after passing through the UV detector, and the
radioactivity in the fractions was measured using an automated gamma
counter.

[0244] The peak corresponding to the intact [18F]B-3 eluted at
˜15 min, whereas the major radiometabolite [18F]I-7 present in
plasma was not detected in brain. The polar metabolite eluting around 2
min was also detected in plasma (vide supra) and passes the
blood-brain-barrier as can be deduced from the in vitro incubation
studies (vide infra). An overview of the results from the perfused rat
brain radiometabolite analysis is presented in Table 17. The fraction of
apolar radiometabolites detected in brain is negligible. The percentage
of polar metabolite detected in cerebellum is higher compared to
cerebrum. At 30 min p.i. about 86% of the recovered radioactivity was
present as intact tracer in cerebrum, in cerebellum this was ˜69%.
After 60 min, the amount of intact tracer in cerebrum was decreased to
˜72%, in cerebellum this was about 47%.

C. Radiometabolite Analysis of Rat Blood, Plasma and Brain Homogenate
after in Vitro Incubation with [18F]B-3 at 37° C. for 60 min

[0245] Blood, plasma and homogenated brain of a rat were incubated with
about 0.17 mCi of [18F]B-3 at 37° C. After 60 min of
incubation, the samples were put on ice to stop the metabolism. Blood and
brain homogenate samples were prepared and analyzed onto RP-HPLC as
described above. The amount of radiometabolites detected in rat blood,
plasma and brain after one hour of incubation was negligable. This
indicates that the polar radiometabolite (hypothesized to be a
[18F]fluoroethyl-derivative) observed in perfused rat cerebrum and
cerebellum at 30 and 60 min p.i. of [18F]B-3, is formed peripherally
(in the liver) and crosses the blood-brain-barrier.

IV. MicroPET Imaging Studies

[0246] Imaging experiments were performed on a Focus® 220 microPET
scanner (Concorde Microsystems, Knoxville, Tenn., USA) using male Wistar
rats with body weight varying between 300 and 600 g. During all scan
sessions, animals were kept under gas anesthesia (2.5% isoflurane in
O2 at 1 L/min flow rate). Dynamic scans of 120 min were acquired in
list mode. After reconstruction of the images, they were
semi-automatically co-registered with a [11C]raclopride template of
the rat brain, and volumes of interest (VOIs) were generated for
different anatomical brain structures (striatum, cerebral cortex and
cerebellum) from which time-activity curves (TAC) were constructed for
each individual image, using PMOD software (PMOD Technologies Ltd.).
Normalization for body weight of the animal and injected dose was done.
The radioactivity concentration in the different brain regions was
expressed as SUV (standardized uptake value) as a function of time in
seconds post injection of the radiotracer. The parametric binding
potential (BP) values were calculated for all acquired images by using a
simplified reference tissue model where the TAC of the cerebellum was
used as input TAC for non-specific binding, using the same software
(PMOD).

A. MicroPET Studies with Compound [18F]B-3

[0247] Three rats were injected with about 2.2 mCi of high specific
activity formulation of [18F]B-3 via tail vein under anesthesia
(2.5% isoflurane in O2 at 1 L/min flow rate) and were scanned
baseline for 2 hours.

[0248] High intensity signal was observed in the striatum with only
background radioactivity in the cortical regions as well as in the
cerebellum. The TACs show that after injection of [18F]B-3 there is
a high initial uptake of the radiotracer in the striatum, cortical
regions and the cerebellum. After this initial high uptake due to the
blood pool activity, the non-specific radioactivity cleared from the
cortical regions and the cerebellum. In striatum, the radioactivity
concentration reached its maximum (average SUV of 1.04) at about 16 min
p.i. and this remained at a similar level until about 53 min p.i. Peak
striatum-to-cerebellum ratios of 5.6 to 1 (n=3) were obtained at 23 min
post injection and these ratios remained around 5.6 until about 53 min
post injection. After this, the ratios decreased slowly until the end of
the experiment due to the clearance of the activity from striatum. The BP
value was calculated by using a simplified reference tissue model where
TAC of the striata was considered as PDE10-rich TAC and TAC of the
cerebellum was used as PDE10 poor TAC. The average BP at baseline was 4.1
(n=3).

[0249] Pretreatment studies where the non-radioactive analogue B-3 was
administered via subcutaneous route at a dose of 2.5 mg/kg body weight
were performed in one rat (one of the three animals that were used in the
baseline scan). At 60 min after pretreatment, the rat was injected with
2.24 mCi of high specific activity formulation of [18F]B-3 via tail
vein under anesthesia (2.5% isoflurane in O2 at 1 L/min flow rate)
and a dynamic scan was performed for 2 hours.

[0250] When the animal was pre-treated with `cold` compound, the
striatum-to-cerebellum ratio decreased to 1.9. Also, there was about 80%
reduction in BP values with pretreatment compared to baseline scans (BP
after blocking=0.8 (n=1)).

[0251] Biodistribution studies as well as microPET imaging studies have
shown specific retention or slower washout of this tracer from the
PDE10-rich region striatum. Therefore, [18F]B-3 is a suitable agent
for imaging and quantification of PDE10A using PET.

B. MicroPET Baseline Study with Compound [18F]B-4

[0252] One rat was injected with about 4 mCi of high specific activity
formulation of [18F]B-4 via tail vein under anesthesia (2.5%
isoflurane in O2 at 1 L/min flow rate) and was scanned baseline for
2 hours.

[0253] High intensity signal was observed in the striatum with only
background radioactivity in the cortical regions as well as in the
cerebellum. The TACs show that after injection of [18F]B-4 there is
a high initial uptake of the radiotracer in the striatum, cortical
regions and the cerebellum. After this initial high uptake due to the
blood pool activity, the non-specific radioactivity cleared from the
cortical regions and the cerebellum. Clearance from striatum was slower,
indicating retention of the tracer in this region. Peak
striatum-to-cerebellum ratios of 4.4 to 1 were obtained at about 6 min
post injection and these ratios remained around 4.4 until about 13 min
post injection. After this, the ratio decreased rather fast due to the
clearance of the activity from striatum. The BP value was calculated from
these images by using a simplified reference tissue model where the
cerebellum was used as a reference tissue. The BP at baseline was 2.5
(n=1).

[0254] Biodistribution studies as well as microPET imaging studies have
shown specific retention or slower washout of [18F]B-4 from the
PDE10-rich region striatum. Compound [18F]B-4 might also be a
suitable agent for imaging of PDE10A expression in the brain using PET.

[0255] Comparison of the kinetics of [18F]B-3 and [18F]Ref The
kinetics of two promising PDE10A PET ligands [18F]B-3 and
[18F]Ref
(2-{4-[1-(2-[18F]fluoroethyl)-4-pyridin-4-yl-1H-pyrazol-3-yl]-phenox-
ymethyl}-quinoline WO-2010/097367) was compared.

[0256] Normal male Wistar rats were injected i.v. via the tail vein under
anesthesia (2.5% isoflurane in O2 at 1 L/min flow rate) with high
specific activity tracer [18F]B-3 or [18F]Ref and scanned
dynamically using μPET for 120 min. During the scan sessions, the
animals were kept under gas anesthesia (2.5% isoflurane in O2 at 1
L/min flow rate). The radioactivity concentration in the different brain
regions was expressed as SUV value as a function of time p.i. of the
radiotracer by normalization for body weight of the animal and injected
dose.

Baseline Time-Activity Curves

[0257] For both tracers, there is a clear wash-out of radioactivity from
cerebellum and cortex, brain regions where the expression of PDE10A is
minimal Both tracers are retained in striatum, however the kinetics in
this brain region is different for both PET ligands. [18F]Ref
reaches its maximum radioactivity concentration [average SUV of 0.73] at
about 57 min p.i. and this stays at the same level until the end of the
experiment. [18F]B-3 reaches its maximum radioactivity concentration
[average SUV of 1.04] at about 16 min p.i. Wash-out of [18F]B-3 from
striatum starts at about 53 min p.i. The faster kinetics of [18F]B-3
compared to [18F]Ref is also reflected in the striatum-to-cerebellum
ratios. For [18F]B-3 peak striatum-to-cerebellum ratios of about 5.6
were obtained at 23 min p.i. and these ratios remained around 5.6 until
about 53 min p.i. After this, the ratios decreased slowly until the end
of the experiment due to the clearance of the activity from striatum. For
[18F]Ref a maximum striatum-to-cerebellum ratio of 4.2 was reached
after about 32 min p.i., which stayed constant until the end of the scan.